The present invention describes the nucleotide sequence of the human homologue of a yeast helicase, pif1, the amino acid sequence of the protein, and uses of the helicase. Preferably, the invention comprises a human Pif-1 type helicase.
Normal human somatic cells (e.g., fibroblasts, endothelial, and epithelial cells) display a finite replicative capacity of 50-100 population doublings characterized by a cessation of proliferation in spite of the presence of abundant growth factors. This cessation of replication in vitro is variously referred to as cellular senescence or cellular aging. The replicative life span of cells is inversely proportional to the in vivo age of the donor, therefore, cellular senescence is suggested to play an important role in aging in vivo.
Cellular immortalization (the acquisition of unlimited replicative capacity) may be thought of as an abnormal escape from cellular senescence. Shay et al., Exp. Cell Res. (1991) 196:33. Normal human somatic cells appear to be mortal, i.e., have finite replicative potential. In contrast, the germ line and malignant tumor cells are immortal (have indefinite proliferative potential). Human cells cultured in vitro appear to require the aid of transforming viral oncoproteins and telomerase overexpression to become immortal (Hahn et al., Nature 400:464-468,1999).
DNA at chromosome ends is maintained in a dynamic balance of loss and addition of telomeric simple sequence repeats. Sequence loss occurs during cell replication, in part from incomplete replication of chromosome termini by DNA-dependent DNA polymerase. Telomeric repeat addition is catalyzed by the enzyme telomerase: a ribonucleoprotein enzyme which uses a short region within the RNA as a template for the polymerase reaction. Although cells can maintain a constant number of telomeric repeats by balancing loss and addition, not all cells do so. Human germline and cancer cells maintain a constant number of telomeric repeats, while normal human somatic cells lose telomeric repeats with each cycle of cell division. As described above, cells that do not maintain stable telomere length demonstrate a limited proliferative capacity; these cells senesce after a number of population doublings correlated with the erosion of telomeres to a critical minimum length.
Because normal somatic cells do not appear to express or require telomerase and do not maintain chromosome ends, and because all or almost all cancer cells express high levels of telomerase activity and maintain chromosome ends, molecules that inhibit or alter telomerase activity could provide effective and non-toxic anti-cancer agents. Similarly, inhibition of telomerase in parasitic or infectious agents (e.g., trypanosomes, fungi, etc.) could provide a specific approach for reducing the viability or proliferation of these agents. Conversely, activation of telomerase in proliferation-restricted cells (such as normal somatic cells, e.g., of the blood, vasculature, liver, skin, etc.) could provide a mechanism for promoting additional proliferative lifespan (i.e., avoid cellular senescence, Hahn et al, 1999). For a review of telomerase and its function, see U.S. Pat. No. 5,770,422 incorporated herein by reference in its entirety.
Pif-1 helicase has been identified in the yeast Saccharomyces as a required participant of both de novo telomere formation and telomere elongation. U.S. Pat. No. 5,466,576 (incorporated by reference herein in its entirety). Pif-1 helicase works by controlling the activity of telomerase and/or interaction with components of the replication machinery. Deletion mutations of either yeast pif1 or its closely related antagonic pif-like helicase RRM3 affects the ability of the cells to replicate and to maintain their normal telomere length(i.e., helicase is related to cell senescence). The recent discovery of loop structures at telomeric DNA ends suggests an important role for helicases in telomere maintenance and replication (Griffith et al., Cell97:503-514, 1999)
A need exists in the art to know the nucleic acid sequence of human pif-1 helicase, in order to effectively screen for [small molecules] targets which modulate helicase activity. Such compounds that modulate helicase activity are useful in two ways: (1) By decreasing the activity of human helicase, the level of telomerase activity or the size of telomeres will be reduced and the viability of the cell is reduced (i.e., replication delayed, slowed down or arrested); and (2) by increasing the helicase activity within a cell, the activity of the telomerase or the length of telomeres may be increased and the viability of the cell increased (i.e., avoid cellular senescence). Applicants, therefore, provide herein for the first time the nucleic acid and amino acid sequence of human Pif-1 type helicase, the only mammalian helicase involved in telomere maintenance described up to now.
Applicants provide herein the nucleic acid sequence of human Pif-1 helicase. Also provided in the amino acid sequence of human Pif-1 helicase, as well as methods for screening for compounds that are capable of modulating the activity of human helicase. Thus, the present invention therefore provides a purified and isolated nucleic acid molecule, preferably a DNA molecule, having a sequence which codes for a human helicase, or an oligonucleotide fragment of the nucleic acid molecule which is unique to the human Pif-1 helicase of the invention and include host cells and expression vectors useful in the expression of human Pif-1 helicase. In a preferred embodiment of the invention, the purified and isolated nucleic acid molecule has the sequence as shown in SEQ ID NO:1.
The invention also contemplates a double stranded nucleic acid molecule comprising a nucleic acid molecule of the invention or an oligonucleotide fragment thereof hydrogen bonded to a complementary nucleotide base sequence.
The present invention also provides: (a) a purified and isolated nucleic acid molecule comprising a sequence as shown in SEQ ID NO:1; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences having at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions. In particular, those sequences containing conserved motifs characteristic of helicases.
The present invention also relates to methods of affecting the viability of a cell or cells by contacting the cell or cells with a modulator of the activity of human Pif-1 helicase in the cell. Such contacting specifically increases or decreases the activity of the helicase in that cell or cells, and therefore the viability of the cell or cells. Preferably such modulators are specific inhibitors of human Pif-1 helicase. Preferably, such modulators are small chemically defined molecules or other polypeptides affecting the helicase activity.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. All references cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.